U.S. patent number 5,206,122 [Application Number 07/662,586] was granted by the patent office on 1993-04-27 for light stable physical developer.
This patent grant is currently assigned to Janssen Pharmaceutica N.V.. Invention is credited to Marcus J. M. Noppe, Lucas A. M. Van Nuffel.
United States Patent |
5,206,122 |
Noppe , et al. |
April 27, 1993 |
Light stable physical developer
Abstract
A light-stable physical developer comprising a solution of
silver ions, a desensitizing agent and a reducing agent. A method
for the detection of one or more components of an aggregate formed
between at least one specific binding agent and its corresponding
bindable substance by labelling at least one component of said
aggregate with a marker and contacting said aggregate with said
light-stable physical developer, whereby under influence of the
marker a metal particle is formed which can be detected. Further
the invention also relates to products, e.g. a test-kit adapted for
carrying out the above mentioned method.
Inventors: |
Noppe; Marcus J. M. (Kalmthout,
BE), Van Nuffel; Lucas A. M. (Merksem,
BE) |
Assignee: |
Janssen Pharmaceutica N.V.
(Beerse, BE)
|
Family
ID: |
10672621 |
Appl.
No.: |
07/662,586 |
Filed: |
February 28, 1991 |
Foreign Application Priority Data
|
|
|
|
|
Mar 14, 1990 [GB] |
|
|
9005753 |
|
Current U.S.
Class: |
430/414; 430/413;
430/477; 430/424; 430/417 |
Current CPC
Class: |
G01N
33/585 (20130101); G03C 5/58 (20130101) |
Current International
Class: |
G03C
5/58 (20060101); G01N 33/58 (20060101); G03C
005/24 () |
Field of
Search: |
;430/413,414,417,424,477 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Le; Hoa Van
Claims
We claim:
1. A physical developer comprising an aqueous solution of silver
ions, a densensitizing agent and a reducing agent.
2. A physical developer according to claim 1 wherein the
desensitizing agent is
6-ethoxy-1-methyl-2-(3-nitrostyryl)quinolinium methyl
sulfonate.
3. A physical developer according to claim 1 comprising a molar
excess of complexant with respect to the silver ions.
4. A physical developer according to claim 3 wherein the complexant
is histidine, imidazole, benzimidazole, pyrazole, pyridine,
aminopyridine, nicotinamide, quinoline or purine.
5. A physical developer according to claim 1 wherein the reducing
agent is 1,2-dihydroxybenzene, 1,4-dihydroxbenzene,
4-methylaminophenolsulfate, 4-aminophenol, 1,4-diaminobenzene,
1,2-diaminobenzene, N-(4-hydroxyphenyl)glycine, 2,4-diaminophenol,
1-phenyl-3-hydroxypyrazole or a mixture thereof.
6. A physical developer according to claim 1 obtained by mixing
a) an aqueous solution comprising a reducing agent and optionally a
buffer system and one or more adjuvants; and
b) an aqueous solution comprising silver ions, a densensitizing
agent, a molar excess of complexant with respect to the silver ions
and optionally a buffer system and one or more adjuvants.
Description
BACKGROUND OF THE INVENTION
During the last years, methods have been introduced wherein
aggregates formed between specific binding agents and bindable
substances are detected by labelling said aggregates directly or
indirectly with small size metal particles, particularly gold
particles. Depending on the circumstances, these particles can be
detected, e.g. by direct visual examination, by microscopic or
spectrophotometric techniques. A description of the "immunogold
staining (IGS) technique", "the sol particle immuno assay (SPIA)
technique" or specific applications and improvements thereof can be
found in U.S. Pat. Nos. 4,313,734, 4,446,238, 4,420,558,
EP-A-0,165,633, EP-A-0,165,634, EP-A-0,158,746, EP-A-0,293,947 and
in IBRO handbook series, Wiley, New York, 1983, pages 347 to
372.
Starting from a relatively unknown method for labelling cell
surface antigens, metal particles have today become widely used in
a variety of detection and/or quantitive determination problems.
The possibility of direct visual examination of metal particles and
the advantage that the signal generated is permanent and not prone
to rapid degradation makes it an interesting marker for simple and
rapid assays. Moreover metal markers, preferably gold markers, seem
preferable over radioisotope markers due to the very low health
hazard related to working with the former.
Next it was found that the signal of a metal marker such as
colloidal gold can be augmented significantly by subjecting the
colloidal gold markers to a so-called physical enhancement
procedure. The effect of such a physical enhancement procedure is
that the typical reddish optical gold signal turns into a
deep-brown to black silver signal having a much higher intensity.
In said procedure, the metal markers used as a label catalyze the
reduction of silver ions present in the developing solution. The
latter results in a specific deposition of a metallic silver layer
at the metal particle site. The thus formed metallic silver
particles in turn catalyze the reduction of more silver ions from
the physical developing solution, creating an autocatalytic
process.
Art-known physical developing solutions generally consist of a
solution containing a soluble metal salt such as silver nitrate, a
reducing agent such as hydroquinone, an appropriate buffer and
optionally a complexant to tie up the metal ions and make them less
susceptible to reduction.
Although the use of these art-known physical developers results in
an augmented signal, there are a number of drawbacks associated
with it. Indeed, it is well known that silver ions may form light
sensitive salts such as silver bromide and silver chloride which
are readily reduced to metallic silver under the influence of
light, starting an autocatalytic process. This non-specific process
of self-nucleation contributes to the occurence of background to a
significant extent. It may be disturbingly strong under conditions
of intense light, e.g. in the monitoring of test samples under a
light microscope, or also when the physical development process is
slow and the test sample is exposed to light for a prolonged
time.
Consequently, the aim of the present invention is to provide a
sensitive and practicable physical developer for use in a variety
of metal based assays which is light-stable and does not give rise
to undesired non-specific deposition of metal particles.
The present invention relates to a physical developer comprising an
aqueous solution of silver ions, a reducing agent, a desensitizing
agent and, if desired, a buffer system and one or more
adjuvants.
Further there is provided a method for qualitatively and/or
quantitatively determining one or more components of an aggregate
formed between at least one specific binding agent and its
corresponding bindable substance, which comprises labelling at
least one component of said aggregate with a marker and contacting
said aggregate with a physical developer whereby under influence of
the marker a silver particle is formed which can be determined
qualitatively, characterized in that the physical developer is a
physical developer according to the present invention, comprising a
solution of silver ions, a desensitizing agent and a reducing
agent.
Another aspect of the present invention is to provide versatile
products such as test-kits adapted for carrying out the above
mentioned methods.
In the present invention the light-sensitivity of the traditional
physical developers based on silver ions is counteracted by adding
a desensitizing agent to the developer. Preferred desensitizing
agents for use in the present invention are electron acceptors such
as, 6-ethoxy-1-methyl-2-(3-nitrostyryl)quinolinium methyl sulfonate
known as Pinakryptol Yellow.RTM. and the like desensitizing
agents.
The reducing agents for use in the present physical developer
include any agents which reduce silver ions from a physical
developer in proximity of an active site. Preferably said reducing
agents form stable solutions with the constituents of the improved
physical developer. As a reducing agent there may particularly be
mentioned, 1,2-dihydroxybenzene, 1,4-dihydroxybenzene
(Hydroquinone), 4-methylaminophenolsulfate (Metol.RTM.),
4-aminophenol, 1,4-diaminobenzene, 1,2-diaminobenzene,
N-(4-hydroxyphenyl)glycine, 2,4-diaminophenol,
1-phenyl-3-hydroxypyrazole (Phenidone.RTM.) or mixtures thereof. As
other adjuvants of the improved physical developer there may be
mentioned, buffers, preservatives, e.g., anti-oxidants or organic
stabilizers, speed regulators, bactericides and the like, such as,
for example, sodium sulfite, sodium bisulfite, sodium citrate and
the like.
Suitable pH adjusting agents are for example, acetic acid, citric
acid, sodium hydroxide or a salt of any of these or a buffer system
based on tris(hydroxymethyl)aminomethane. The pH of the physical
developer preferably ranges from about 5 to 9, in particular from
about 6 to 8. In general the pH of the physical developer is
limited to the range wherein the specific binding agent and the
corresponding bindable substance are stable.
Apart from the silver ions, the reductant and desensitizing agent,
the preferred physical developing solution also comprises an excess
of a complexant to tie up the metal ions and to make them less
susceptible for reduction. Favourable complexants for use in the
present invention are described in EP-A-0,293,947 and include
pyridine, aminopyridine, nicotinamide, quinoline, imidazole,
histidine, benzimidazole, pyrazole, purine and the like aromatic
heterocyclic ring systems.
An exemplary mode of preparing the physical developer of this
invention comprises dissolving or suspending the desensitizing
agent in an aqueous solution comprising silver ions, reductant,
buffer and any adjuvants. In the final solution, the ratio of
desensitizing agent:silver ions is from about 50 g/mol to 0.5 g/mol
silver ions, in particular from about 50 g/mol to 5 g/mol silver
ions, or from 35 g/mol to 15 g/mol silver ions. The concentration
of silver ions ranges from 0.001 mol/l to 0.1 mol/l in particular
from about 0.005 mol/l to 0.5 mol/l, or from 0.07 mol/l to 0.3
mol/l.
In a preferred embodiment the physical developer is prepared by
mixing two stable and liquid solutions. One solution, hereinafter
being referred to as enhancer, comprises silver ions, a
desensitizing agent, a molar excess of complexant with respect to
the silver ions and optionally a buffer system. In particular the
molar excess of complexant versus the silver ions ranges from about
a two to a twohunderd-fold molar excess, preferably from about a
twenty to a hunderd-fold molar excess. The other solution,
hereinafter being referred to as initiator, comprises a reducing
agent and optionally a buffer system and an anti-oxidant and/or
organic stabilizer, such as, for example, sodium sulfite, sodium
bisulfite and the like. Preferably the dilution of both the
enhancer and the initiator are such that mixing an equal volume of
each solution will yield a light-stable physical developer as
described hereinabove. In some instances the enhancer and initiator
may be prepared from their corresponding dry constituents by adding
an appropriate amount of water. In order to facilitate the
preparation of the enhancer and/or physical developer it may be
appropriate to dissolve the desensitizing agent first in a small
amount of an organic solvent such as N,N-dimethylformamide,
N,N-dimethylacetamide and the like and to mix to thus obtained
organic solution with the aqueous solution comprising the silver
ions. The pH of the enhancer should be in the range from about 5 to
9, preferably from about 6.5 to 8.5. The pH of the initiator should
be in the range from 2 to 7, preferably from about 3.5 to 5.5.
Although the use of desensitizing agents like Pinakryptol
Yellow.RTM. in silver halide emulsions is known, it is surprising
that Pinakryptol Yellow.RTM. can selectively prevent non-specific
self-nucleation in a physical developing solution and form a light
stable aqueous solution, while not adversely affecting the rate of
metal deposition on the marker. Thus the present invention provides
a severalfold increase in the ratio between marker-specific
reduction rate and rate of self-nucleation. A noteworthy
consequence thereof is that the present physical developer can
react to the maximum, i.e. until depletion of all silver ions,
whereafter both marker specific development and self-nucleation--if
any--sharply decrease and stop. This makes the physical developing
procedure less dependent on external and procedural parameters.
More in particular this means that one does not any longer need to
time the development process in order to stop it when background
noise by non-specific metal deposition starts. With the
compositions of the present invention the development process may
run to completion and therefore be left unmonitored.
The increased ratio between marker-specific reduction rate and rate
of non-specific self-nucleation can be exploited in several ways.
The sensitivity can be increased by keeping the marker longer into
contact with the physical developer, or the speed of the
marker-specific development can be increased and this without
loosing the flexibility offered by a safe period of time between
the moment of optimum marker development and the moment where
self-nucleation starts to give an increased background. In some
cases a combination of both increased overall speed and sensitivity
can be implemented. The speed and sensitivity can be modulated by
changing the concentration and the nature of the silver ions, in
particular their ligands, the desensitizing agent, the reducing
agent and/or the pH of the developing solution. For example,
increasing the concentration of the silver ions and/or increasing
the pH and/or using a stronger reducing agent will result in a
faster developing procedure. Conversely, the developing procedure
can be slowed down by lowering the concentration of silver ions
and/or lowering the pH and/or by employing a weaker reducing agent.
The speed, especially with markers smaller than 5 nm, can perfectly
be modulated from very fast (10-20 sec) to slow (30 min or more).
Preferably the developing time is modulated from about 10 seconds
to two minutes, in particular from 10 seconds to one minute.
The above described physical developers are preferably employed in
methods for qualitatively and/or quantitatively determining one or
more components of an aggregate formed between at least one
specific binding agent and its corresponding bindable substance
whereby at least one component of said aggregate is labelled with a
metal marker which catalyzes directly or indirectly the reduction
of silver ions from a physical developer.
The marker for use in the method according to the invention defines
any particle which can catalyze the reduction of metal ions,
resulting in a deposition of the corresponding metal particles at
the site of the said marker.
Said markers comprise metals, metal compounds or polymers
optionally coated or impregnated with metals or metal compounds
which can catalyze directly or indirectly the reduction of metal
ions on their surface. As examples of such metals there may be
named gold, silver, thallium, platinum, palladium as well as
copper, nickel and the like with gold being preferred. As examples
of metal compounds there may be named their corresponding complexes
or chelates and sulfides. Polymers coated or impregnated with
metals or metal compounds have similar properties as the metal or
metal compounds but size, density and metal content can be
optimally combined. For use in the preferred method the marker
should be selected so that specific binding agents or their
corresponding bindable substances can be attached to the marker,
without loss of their affinity for their counterpart.
Particularly preferred markers for use in the method according to
the present invention are either (i) colloidal metal particles,
optionally a sol containing metals or metal sulfides; or (ii) metal
chelates, especially those incorporating ethylenediaminotetraacetic
acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) groups; or
(iii) polymers optionally impregnated with metals or metal
sulfides, e.g., polymerization products of benzidine derivatives
such as, for example diaminobenzidine polymers.
Said preferred method can conveniently be carried out by
immobilizing the specific binding agent or the corresponding
bindable substance, directly or indirectly, on a solid support,
contacting the support with a counterpart labelled with a marker
which catalyzes the reduction of the metal ions of the physical
developer, and adding the physical developer before or after the
separation of the bound and free labelled components, whereby
during the reaction or after an adequate reaction time, the formed
metal particles are quantitatively and/or qualitatively determined
in the test sample and/or in the derived fractions to provide a
qualitative and/or quantitative indication of the component or
components to be determined. In some instances it may be preferable
to contact the support containing the immobilized bindable
substance with a first binding agent specific to said bindable
substance to form an aggregate herewith, and subsequently
contacting the support carrying the thus formed aggregate with a
second binding protein, which is specific to the said first binding
protein, labelled with marker. The thus described method is
particularly suited for the determination of immunochemical
components, such as haptens, antigens and antibodies.
Further, the present invention may also be employed for
quantitatively and/or qualitatively determining an acceptor
substance, such as a protein or a nucleic acid, which is directly
immobilized on a solid support and bound with the aforementioned
marker.
The determinations to be made according to the preferred method of
this invention may be performed homogeneously or heterogeneously.
Homogeneous determinations are particularly simple to perform but
require a measurable change of the perceived signal arising from
either those markers present in the labelled reagent or in the
labelled aggregate formed between the labelled reagent and the
particles to be determined. In those instances where no such
distinction is possible, heterogeneous determinations will have to
be performed.
Homogeneous determinations are advantageous due to the fact that it
is not necessary to physically separate the bound and unbound
labelled species, thus reducing the number of steps necessary to
perform an assay. The reaction between the labelled component and
the corresponding binding counterpart causes the measurable change
in the label's participation in or modulation of the signal
generating moiety necessary to perform a homogeneous determination.
The distribution of the markers between the bound and unbound
species may be differentiated by the inability or altered ability
of the said markers to affect the signal arising therefrom after
development when present in the bound species.
A homogeneous determination may conveniently be performed according
to art-known procedures such as, for example, the competitive
binding technique. The sample containing the analyte is combined
with a binding counterpart of the analyte, a labelled reagent
comprising a marker coupled to the analyte or a specific binding
analogue thereof, and the physical developer necessary to convert
the marker to the signal generating moiety itself. Alternatively, a
sequential determination may be performed whereby the sample and
the analyte binding counterpart are first combined and thereafter
the detectant reagent added.
In many instances it is not possible to perform homogeneous
determinations. In these cases a heterogeneous determination can be
a particularly attractive alternative. In general, the
heterogeneous determination system comprises at least two basic
constituents and the physical developer which are combined
simultaneously or subsequently, i.e. the analyte to be detected, a
binding counterpart labelled with a marker and the physical
developer necessary to convert the marker to the signal generating
moiety itself. If necessary after an appropriate incubation period
or periods the labelled reagent becomes bound to the corresponding
bindable substance to be detected whereby the ratio of the bound
species to the unbound species is a function of the amount of
analyte being present. The bound and unbound species are physically
separated and the amount of label being present in one thereof is
determined.
Various means of performing the separation step and of performing
the binding reactions are known in the art. The said separation may
involve conventional techniques such as, for example, by employing
a solid-phase antibody or antigen, a second antibody, or a
solid-phase second antibody; or by the use of immuno complex
precipitation agents, adsorbents, and the like. The said binding
reactions may for example include the so-called competitive binding
technique, the sequential saturation technique, the sandwich
technique, and the like.
The preferred determinations to be made according to the method of
this invention are heterogeneous determinations which are generally
based on the principle that the labelled aggregate formed between
the specific binding protein and the bindable substances is at some
time immobilized in such manner that any unreacted particles can be
washed off, whereupon the immobilized particles are detected "in
situ" or, if desired, after disengagement in any other phase
derived therefrom.
In a particularly preferred embodiment, the binding substance to be
detected, which may be contained in a crude test specimen or in a
purified or partly purified fraction derived therefrom, is
immobilized on an appropriate immobilizing support prior to its
complexing with the labelled binding agent, specific to said
bindable substance.
The immobilization of the bindable substance may be carried out
following the usual techniques, e.g., by spotting an aliquot of the
test specimen on the immobilizing support or by immersing the
latter in the test sample and subsequently drying and optionally
washing off non-immobilized material. This is the so-called direct
technique. As immobilizing supports for this technique use can be
made of various materials, in general polymeric materials like,
nitrocellulose, diazobenzyloxymethyl (DBH)- and
diazophenylthioether (DPT) modified cellulose paper, paper, paper
or cellulose acetate activated with cyanogen bromide, agarose,
nylon, plastics and the like, which may take any form which is
convenient for the determination process, e.g. sheets, beads,
welled plates, dip-sticks and the like.
The support is then brought into contact with a labelled binding
agent under conditions which allow aggregate formation between the
binding agent and the corresponding bindable substances.
Consequently, at the sites where the bindable substance is
immobilized, markers will be immobilized in turn in amounts
proportional to the concentration of the immobilized bindable
substance.
In a variant of this method, the immobilized bindable substance is
first allowed to react with a first binding agent which is specific
therefore and subsequently the thus immobilized phase is brought
into contact with the markers attached to a second binding agent
which is specific for said first binding agent.
Because of the lack of selectivity and specificity of the
immobilizing process as described above, the direct method is
usually employed with relatively pure or purified test samples or
fractions. For more complex samples, the direct method will often
be less suitable, as the non-specific immobilization of a large
excess of non-desired material will interfere with the sensitivity
and specificity of the determination.
To avoid this problem, which is important with regard to routine
analyses, an indirect or so-called sandwich technique can be used.
In this technique, a purified or enriched primary specific binding
agent is immobilized on a solid support. The latter is contacted
with the test sample under conditions which allow the complexing of
the corresponding bindable substances, which consequently become
immobilized themselves. After removal of the test sample and
washing of the support, the latter is contacted with a suspension
of markers coated with secondary specific binding agents which are
able to bind to uncomplexed sites of the immobilized bindable
substance.
The most straightforward case of embodiment to which the invention
is applicable is a flow-through environment consisting of bindable
substance which is immobilized, directly or indirectly, to a solid
phase, and a liquid phase mobile relative to the solid phase.
Depending on the direction of the liquid phase flow versus the
solid phase, this solid phase can be liquid-permeable or
-unpermeable. For example, a permeable membrane can be used as
solid phase, allowing for a perpendicular flow of the liquid phase
through that membrane. On the other hand, an unpermeable solid
phase can be used in combination with a lateral liquid flow.
During the first step of the embodiment, the liquid phase
containing the marked specific binding agent is brought into
contact with the bindable substance immobilized on the solid phase.
The movement of this liquid phase relative to the solid phase may
be continuous or discontinuous and must be such that the contact
time between both phases allows for binding between the immobilized
bindable substance and the marked specific binding agent to take
place. However, this binding process not necessarily needs to reach
its saturation point. The pressure drop between source and
destination of the liquid phase, creating its flow, may be built in
several ways. In the case of a permeable membrane as solid phase, a
perpendicular flow may be created by bringing one side of this
membrane into contact with a fluid-absorbing material and by
applying the liquid phase on the other side. In the case of a
non-permeable solid phase, a lateral flow of the liquid phase may
be created by a pump.
For the second step an improved physical developer according to
this invention is applied as the liquid phase. In order to maximize
the ratio between marker-specific reduction speed and the speed of
self-nucleation, it may be appropriate to keep both components of
the physical developer, initiator and enhancer, apart until
immediately before use. However, it should be emphasized that in
comparison with the prior-art, stable solutions of the physical
developer can be made by mixing the initiator and enhancer. In some
instances it may be preferable to apply the enhancer and initiator
subsequently and thus forming the improved physical developer "in
situ". In view of the above considerations the physical developer
of the present invention can easily be optimized towards the marker
used, the sensitivity required and the contact time between the
solid phase and the liquid physical developer.
In the preferred flow-through embodiment, the mixing of the two
stable liquid components and the application of the resulting
improved physical developer should be combined into a single
action. The application of the flowing protected physical developer
has a dual effect. Initially, the liquid will wash away from the
solid phase all remaining marked specific binding agents which were
not or only loosely bound during the first step. Because the
physical development of the marker is a gradually progressive
process, this material will be washed from the solid phase before
any signal becomes apparent. The remaining, bound marked specific
binding agents will generate a visible signal during the further
contact with the protected physical developer. The flow and volume
of the developer applied to the solid phase can be chosen to ensure
that the contact time is long enough for an optimal detection of
the immobilized marked compounds and short enough to avoid
non-specific reduction at the solid phase caused by
self-nucleation. Typically, these times can be modulated from a few
seconds to several minutes.
The detection of the formed metal particles in a certain phase of
the reaction mixture may take place using numerous techniques which
are in themselves known. Said techniques are based upon the amount
and/or the physical properties of the metal particles formed,
preferably on the scattering and adsorption of the metal particles.
As examples of these techniques there may be cited the
spectrophotometric techniques such as densitometry, which will be
preferred when quantitative determinations are desired. However, in
view of the high sensitivity obtained the particles can easily be
observed visually, optionally using a microscope.
The specific binding agents which can be employed in the preferred
method according to the invention can be of various nature but will
in many instances be antibodies to specified antigens or haptens.
As an example of specific binding substances other than antibodies
there can be mentioned phages, which are optionally chemically or
genetically adapted to bind molecular or cellular materials,
lectins, which specifically bind glycoproteins, Staphylococcus
aureus protein A which specifically binds immunoglobulins of
various animal species and DNA or RNA probes for gene
identification. In general any other molecular interaction of
sufficient specificity and affinity can be employed. Antibodies may
be polyclonal or monoclonal.
In view of their general nature, the methods according to the
invention have an extremely wide field of application. In principle
they can be applied to the qualitative and/or quantitative
determination of any substance which can be labelled with the
aforementioned marker. For example, such substances comprise but
are not limited to cell surface and tissue antigens, biological
substances excreted by or derived from living organisms,
particularly biological substances occurring in biological fluids
such as saliva, lymph, blood and its derived fractions such as,
plasma and serum, urine, cerebrospinal fluid, amnion fluid, and the
like. Substances which can be detected include, proteins,
polypeptides, peptides, like enzymes, hormones, structural
proteins, nucleic acids, vitamins, polysaccharides, toxins,
alkaloids, glycoproteins, haptens, metabolites, pharmacological
agents, pesticides, pollutants, steroids, and any other molecule
for which a specific binding counterpart exists in biological
systems or can be synthesized.
Representative protein analytes include the classes of protamines,
mucoproteins, glycoproteins, globulins, albumins, scleroproteins,
phosphoproteins, histones, lipoproteins, chromoproteins, and
nucleoproteins. Examples of specific proteins are prealbumin,
.alpha..sub.1 -lipoprotein, human serum albumin, .alpha..sub.1
-acid glycoprotein, .alpha..sub.1 -antitrypsin, .alpha..sub.1
-glycoprotein, transcortin, thyroxine binding globulin,
haptoglobin, hemoglobin, myoglobin, ceruloplasmin, .alpha..sub.2
-lipoprotein, .alpha..sub.2 -macroglobulin, .beta.-lipoprotein,
erythropoietin, transferin, hemopexin, fibrinogen, the
immunoglobulins such as IgG, IgM, IgA, IgD, and IgE, IgG being
preferred and their fragments, e.g., F.sub.c, F.sub.ab and
F(ab).sup.2 complement factors, prolactin, blood clotting factors
such as fibrinogen, thrombin and so forth, insulin, melanotropin,
somatotropin, thyrotropin, follicle stimulating hormone,
luteinizing hormone, gonadotropin, thyroid stimulating hormone,
placental lactogen, intrinsic factor, transcobalamin, serum enzymes
such as alkaline phosphatase, lactic dehydrogenase, amylase,
lipase, phosphatases, cholinesterase, glutamic oxaloacetic
transaminase, glutamic pyruvic transaminase, and uropepsin,
endorphins, enkephalins, protamine, tissue antigens, bacterial
antigens, and viral antigens such as hepatitis associated antigens
(e.g., HB.sub.s Ag, HB.sub.c Ag and HB.sub.e Ag).
Representative hapten analytes include the general classes of
drugs, metabolites, hormones, pesticides, pollutants, vitamins, and
the like organic compounds. Haptenic hormones include thyroxine and
triiodothyronine. Vitamins include vitamins A, B, e.g. B.sub.12, C,
D, E and K, folic acid and thiamine. Drugs include antibiotics such
as aminoglycosides, e.g., gentamicin, tobramycin, amidacin,
sisomicin, kanamycin, and netilmicin, penicillin, tetracycline,
terramycin, chloromycetin, and actinomycetin; nucleosides and
nucleotides such as adenosine diphosphate (ADP) adenosine
triphosphate (ATP), flavin mononucleotide (FMN), nicotinamide
adenine dinucleotide (NAD) and its phosphate derivative (NADP),
thymidine, guanosine and adenosine; prostaglandins; steroids such
as the oestrogens, e.g., oestriol and oestradiol, steroids; and
others such as phenobarbital, phenytoin, pirimidone, ethosuximide,
carbamazepine, valproate, theophylline, caffeine, propranolol,
procainamide, quinidine, amitryptiline, cortisol, desipramine,
disopyramide, doxepin, doxorubicin, nortryptiline, methotrexate,
imipramine, lidocaine, N-acetyl-procainamide, amphetamines,
catecholamines, and antihistamines. Further cardiac glycosides, and
derivatives of benzodiazepine, benzimidazole, piperidine,
piperazine, imidazole, triazole, pyridazine, 1,2,4-triazinedione or
2,3,5,6-tetrahydroimidazo[2,1-b]thiazoles, or amides, hydratropic
acid derivatives or trialkylamines. Benzimidazole haptens comprise
thiabendazole, fuberidazole, ciclobendazole, oxibendazole,
parbendazole, cambendazole, mebendazole, fenbendazole,
flubendazole, albendazole, oxfendazole, nocodazole and astemizole.
Piperidine haptens comprise diphenoxylate, phenoperidine,
haloperidol, haloperidol decanoate, bromperidol decanoate,
bromperidol, moperone, trifluperidol, pipamperone, piritramide,
fentanyl, benperidol, droperidol, benzitramide, benzetimide,
domperidone, sufentanil, carfentanil, alfentanil, dexetimide,
milenperone, difenoxin, fluspirilene, penfluridol, pimozide,
lorcainide, loperamide, astemizole, ketanserine, levocabastine,
cisapride, altanserin, ritanserin,
3-[2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-2,7-dimethyl-4H-pyrido-[1,2
-a]-pyrimidin-4-one,
3-[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2-methyl-4H-py
rido[1,2-a]pyrimidin-4-one and
3-[2-[4-[[3-(2-furanylmethyl)-3H-imidazo[4,5-b]pyridin-2-yl]amino]-1-piper
dinyl]ethyl]-2-methyl-4H-pyrido[1,2-a]pyrimidin-4-one. Piperazine
haptens include azaperone, fluanisone, lidoflazine, flunarizine,
mianserine, oxatomide, mioflazine, clocinizine and cinnarizine.
Examples of imidazole haptens are metronidazole, ornidazole,
ipronidazole, tinidazole, isoconazole, nimorazole, miconazole,
burimamide, metiamide, metomidate, enilconazole or imazalil,
etomidate, econazole, clotrimazole, carnidazole, cimetidine,
doconazole, sulconazole, parconazole, orconazole, butoconazole,
triadiminole, tioconazole, valconazole, fluotrimazole,
ketoconazole, oxiconazole, lombazole, bifonazole, oxmetidine,
fenticonazole, fluconazole, tubulazole and
(Z)-1-[2-chloro-2-(2,4-dichlorophenyl)ethenyl]-1H-imidazole.
Triazole haptens comprise virazole, azaconazole, etaconazole,
propiconazole, penconazole, itraconazole and terconazole.
Pyridazine haptens comprise for example,
3-chloro-6-[3,6-dihydro-4-(3-methylphenyl)-1(2H)-pyridazine,
3-methoxy-6-[4-(3-methylphenyl)-1-piperazinyl]pyridazine and the
compounds of Publ. Eur. Pat. Appl. No. 0,156,433.
1,2,4-Triazinediones comprise for example,
2-chloro-.alpha.-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2
(3H)-yl)benzeneacetonitrile,
2,6-dichloro-.alpha.-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triaz
in-2(3H)-yl)benzeneacetonitrile and the compounds of Publ. Eur.
Pat. Appl. No. 0,170,316. Trialkylamines are, for example,
diisopromine, prozapine. 2,3,5,6-Tetrahydro-imidazo[2,1-b]thiazoles
comprise, for example, tetramisole or levamisole.
Amides comprise for example, closantel, ambucetamide, isopropamide,
buzepide metiodide, dextromoramide. A hydratropic acid hapten is,
for example, suprofen.
The purposes of the determinations can be multiple. In certain
applications the physical developer according to the present
invention will be used merely as a scientific tool in light and
electron microscopic applications, to better visualize particular
substances, e.g. on histological coupes, on chromatograms,
electrophoretograms, blots, etc. It is found that the present
physical developer is especially useful for the immunological
detection of antigens both in cell and tissue sections and in whole
mount (intact cell) specimens. The achieved sensitivities are very
high and the resulting labelling pattern is more homogeneous than
with art-known physical developers, especially when ultra small
markers are applied, such as colloidal gold particles of about 1 to
3 nm. Apart from its scientific utility, the physical developer
according to the present invention will find utility in a wide
variety of diagnostic tests such as, for example, the following:
the detection and characterisation of subpopulations of
T-lymphocytes; pregnancy tests based on the presence of certain
hormones (chorionic gonadotropin) in the urine, diagnostic tests
for various infectious diseases of fungal or bacterial origin, e.g.
gonorrhoea, and in particular of for diseases of viral origin, such
as, for example, hepatitis B, rubella, poliomyelitis and the like,
auto-immune-diseases e.g. Lupus erythematosus and immune deficiency
diseases, e.g. AIDS; diagnostics for metabolic, endocrinological
and various endogenous diseases, including diagnostics for the
detection of congenital malfunctions of embryos based on the
presence of specific proteins in the amnion fluid.
Hence it can be employed in virtually all circumstances for which
immunological techniques are conceived at present. In addition the
physical developer according to the present invention may also be
employed in the determination and/or detection of acceptor
substances, such as proteins or nucleic acids, which are directly
immobilized in or on a solid support and bound with a colloidal
marker following procedures described in the European Patent
Publication No. 0,165,633 and Analytical Biochemistry 145, 315-321
(1985). Said method comprises the subsequent steps of contacting a
protein or nucleic acid support for a given time, with a sufficient
concentration of colloidal markers suspended in a medium,
preferably containing a detergent that does not interfere with
protein or nucleic acid binding, like for example 0.1% of the
non-ionic detergent Tween 20, and appropriately pH adjusted, and
adding a physical developer whereby during the reaction or after an
adequate reaction time the formed metal particles are
quantitatively and/or qualitatively determined. The physical
developer according to this invention improves the sensitivity of
this method without the drawbacks associated with the traditional
developers.
The method according to the invention offer a framework which can
be used for a wide variety of routine and experimental
applications. Due to their nature and ease of handling, the methods
lend themselves particularly for simple and rapid qualitative or
semi-quantitative assays. These can be oriented towards use by
experienced laboratory technicians as well as by non-technically
trained medical trained personnel or laymen. The methods can also
be easily automated which is an important factor when large numbers
of identical determinations must be carried out, e.g., in blood
banks and specialized clinical laboratories.
In further aspect of the invention there are provided products for
depositing silver particles on a marker which catalyzes the
reduction of silver ions from a physical developer. Said products
comprise some or all of the ingredients required to conduct assay
methods as described hereinabove. Said products can be presented in
a commercially packaged form, e.g. as a composition or admixture
when the ingredients are compatible, in a test device
configuration, or as a test-kit, i.e. a packaged combination of two
or more containers holding the necessary ingredients. In its
simplest configuration said product consists of a physical
developer solution as described hereinbefore. Preferably said
products are presented as test kits comprising an enhancer solution
and an initiator solution which upon mixing of two equal volumes of
each solution yield a light-stable physical developer as described
hereinbefore. Said test-kits may further comprise ingredients
appropriate for specific assay methods such as specific binding
agents labelled with metal markers which catalyze the reduction of
silver ions from the physical developer, non-labelled binding
agents specific to analytes together with labelled binding agents
specific to said non-labelled binding agents. Obviously, the
present products may also include other useful reagents such as
buffers, diluents, standards and the like.
EXAMPLES
Example 1
Materials and methods
1.1. Preparation of a protected physical developer I
Two liquid components of this developer were prepared separately.
Solution A was made by dissolving 17.85 g citric acid, 7.05 g
sodium citrate, 40 g imidazole and 1.86 g silver nitrate into 500
ml water. Solution B consists of 32.9 g sodium citrate, 10 g sodium
sulphite, 0.6 g N-(p-hydroxyphenyl)glycine and 15.32 g citric acid
into 1000 ml water.
1.2. Preparation of a protected developer II
Two liquid components of this developer were prepared separately.
Solution A was made by dissolving 17.85 g citric acid, 7.05 g
sodium citrate, 40 g imidazole, 1.86 g silver nitrate and 0.25 g
Pinakryptol Yellow.RTM. dissolved into about 5 ml of
N,N-dimethyl-formamide. The solution was diluted with water to 500
ml. Solution B consists of 32.9 g sodium citrate, 10 g sodium
sulphite, 0.6 g N-(p-hydroxyphenyl) glycine and 15.32 g citric acid
into 1000 ml water.
1.3. Method for measuring selfnucleation
The reagents are brought to 20.degree. C. by putting them in a
thermostated waterbath at 20.degree. C. 1 ml of solution A and 1 ml
of solution B are mixed in a plastic cuvette and put in a
spectrophotometer thermostated at 20.degree. C. The increase in the
optical density at 500 nm is monitored and registered
continuously.
1.4. Visual inspection of selfnucleation
A visual control test for silver nucleation is carried out by
mixing 1 ml of solution A and 1 ml of solution B in a transparent
polystyrene test tubel The time is registered when the first
turbidity is seen. This test can be done in the dark and in the
light conditions.
Results
1. Measuring selfnucleation
The test for measuring the selfnucleation was executed using the
test described in 1.3 using the protected physical developer I and
II described in 1.1 and 1.2. This test shows the selfnucleation
time in absence of light. The results are shown in table 1 and 2.
As shown, there is no difference in the selfnucleation in the dark,
using the protected physical developer I and II.
TABLE 1 ______________________________________ self-nucleation in
dark for physical developer I Times (minutes) Optical density at
500 nm ______________________________________ 0 0.000 2 0.000 4
0.001 6 0.001 8 0.001 10 0.000 12 0.000 14 0.000 16 0.000 18 0.000
20 0.000 ______________________________________
TABLE 2 ______________________________________ self-nucleation in
dark for physical developer II Times (minutes) Optical density at
500 nm ______________________________________ 0 0.000 2 0.000 4
0.000 6 0.000 8 0.000 10 0.000 12 0.000 14 0.000 16 0.000 18 0.000
20 0.000 ______________________________________
2. Visual inspection of selfnucleation
The test for visual control of the selfnucleation was executed
using the test described in 1.4 using the protected physical
developers I and II described in 1.1 and 1.2. The results of this
test is summarized in table 3.
TABLE 3 ______________________________________ physical developer I
physical developer II ______________________________________ First
selfnucleation 6 minutes 27 minutes in full light First
selfnucleation >120 minutes >120 minutes in dark
______________________________________
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